Various publications, including patents, published applications, technical articles and scholarly articles are cited throughout the specification. Each of these cited publications is incorporated by reference herein, in its entirety.
While cell transplantation is a promising therapy to overcome injury and disease, the widespread application of this approach is limited by the poor survival, differentiation, and integration of cells transplanted into a site of injury. Injectable, bioresorbable hydrogels present a viable approach to overcome the barriers of cell survival and integration. Unlike pre-formed tissue-engineered scaffolds that generally require invasive surgical techniques for implantation, injectable hydrogels offer the potential of minimally invasive cell delivery through a needle or catheter into a cavity that results from tissue loss due to disease, aging or injury, or into the healthy tissue near the diseased or injured or aged tissue. The gel could conform to the shape of the lesion cavity if injected therein where it could also serve as a “bridge” across which regeneration may occur. Most of the hydrogel systems developed to date have been designed for the delivery of therapeutic molecules. This is especially true to the injured or diseased central nervous system (CNS), where neurotrophic factors [Tomita M, Lavik E, Klassen H, Zahir T, Langer R, Young M J. Biodegradable polymer composite grafts promote the survival and differentiation of retinal progenitor cells. Stem Cells 2005; 23:1579-88; Lavik E B, Klassen H, Warfvinge K, Langer R, Young M J. Fabrication of degradable polymer scaffolds to direct the integration and differentiation of retinal progenitors. Biomaterials 2005; 26:3187-96; Kubitz J C, Motsch J. Eye surgery in the elderly. Best Pract Res Clin Anaesthesiol 2003; 17:245-57; Tschon M, Fini M, Giavaresi G, Torricelli P, Rimondini L, Ambrosio L et al. In vitro and in vivo behaviour of biodegradable and injectable PLA/PGA copolymers related to different matrices. Int J Artif Organs 2007; 30:352-62], neuroprotective molecules [Wallace D G, Rosenblatt J. Collagen gel systems for sustained delivery and tissue engineering. Advanced Drug Delivery Reviews 2003; 55:1631-49], and antibodies against axonal regeneration inhibitors [Chemte A, Chaput C, Wang D, Combes C, Buschmann M D, Hoemann C D et al. Novel injectable neutral solutions of chitosan form biodegradable gels in situ. Biomaterials 2000; 21:2155-61] have been delivered. The necessity to enhance the survival of transplanted cells (primary, cell lines, stem/progenitor or precursor cells) in vivo requires a delivery vehicle. This is important to numerous diseases and injuries, including diabetes, heart-related conditions, arthritis (osteo and rheumatoid), joint injuries, CNS diseases and disorders including retinitis pigmentosa, age-related macular degeneration, Parkinson's disease, Huntington's disease, Alzheimer's disease, multiple sclerosis, amyotrophic lateral sclerosis, spinal muscular atrophy, age-related macular degeneration, stroke, cerebral palsy, and spinal cord injury.
Diseases of the retina and retinal function can lead to permanent loss of visual function for which there is no definitive treatment. The detrimental impact of vision loss on quality of life and activities of daily living has been well documented and affects the entire age spectrum. Retinitis pigmentosa (RP) affects the pediatric and young adult population, and is the leading cause of inherited retinal degeneration-associated blindness [Shintani K, Shechtman D L, Durwood A S. Review and update: Current treatment trends for patients with retinitis pigmentosa. Optometry 2009; 80:384-401]. Diabetic retinopathy is the principle cause of blindness in middle-aged working adults [Klein B E K. Overview of epidemiologic studies of diabetic retinopathy. Ophthalmic Epidemiol 2007; 14:179-83]. Age-related macular degeneration (AMD) is the leading cause of irreversible blindness and moderate visual impairment in developed nations: there are an estimated 200,000 new cases annually in the United States [Kaufman S R. Developments in age-related macular degeneration: Diagnosis and treatment. Geriatrics 2009; 64:16-19]. Irreversible photoreceptor death or loss of function is common to all of these pathologies. It is expected that rates of blindness due to retinal degeneration will rise as our population ages in the coming decades [Congdon N G, Friedman D S, Lietman T. Important Causes of Visual Impairment in the World Today. J Am Med Assoc 2003; 290:2057-6; Lee P, Wang C C, Adamis A P. Ocular neovascularization: An epidemiologic review. Surv Ophthalmol 1998; 43:245-69], providing a strong impetus in the search for new therapies.
Current therapies for vision loss have focused predominantly on pharmacological treatments. For example, there have been recent advances in the treatment of the neovascular (wet) form of AMD with anti-vascular endothelial growth factor therapies [Rosenfeld P J, Brown D M, Heier J S, Boyer D S, Kaiser P K, Chung C Y et al. Ranibizumab for neovascular age-related macular degeneration. N Engl J Med 2006; 355:1419-31; Menon G, Walters G. New paradigms in the treatment of wet AMD: The impact of anti-VEGF therapy. Eye 2009; 23(suppl. 1):S1-7]. Experimental treatments of diabetic retinopathy focus on bioactive molecules such as advanced glycosylation end product inhibitors and anti-oxidants to counter oxygen-induced injury [Corner G M, Ciulla T A. Current and future pharmacological intervention for diabetic retinopathy. Expert Opin Emerg Drugs 2005; 10:441-55]. While these therapies show promise in limiting the pathophysiologic advancement of the disease, they do not represent a restorative approach.
Cellular transplantation therapy is an alternate strategy in which auto- or allogenic cellular material is used to replenish damaged retinal cells. The inner retinal microstructure in both AMD and RP is relatively intact following pathological photoreceptor degeneration, and one regenerative approach is to repopulate these cells without having to recapitulate the intricate retinal architecture. Various types of retinal tissue have now been allografted in the treatment of retinal disease: fetal retinal pigmented epithelium (RPE) cells to patients with AMD [(9) Algvere P V, Gouras P, Kopp E D. Long-term outcome of RPE allografts in nonimmunosuppressed patients with AMD. Eur J Ophthalmol 1999; 9:217-30; Algvere P V, Berglin L, Gouras P, Sheng Y. Human fetal RPE transplants in Age Related Macular Degeneration (ARMD). Invest Ophthalmol Vis Sci 1996; 37:S96], and neural retinal cells to patients with RP [Das T P, Del Cerro M, Lazar E S, Jalali S, DiLoreto D A, Little C W et al. Transplantation of neural retina in patients with retinitis pigmentosa. Invest Ophthalmol V is Sci 1996; 37:S96]. Treating AMD by targeting RPE regeneration or transplantation is a therapeutically relevant option being pursued through research [Chen F K, Uppal G S, Maclaren R E, Coffey P J, Rubin G S, Tufail A et al. Long-term visual and microperimetry outcomes following autologous retinal pigment epithelium choroid graft for neovascular age-related macular degeneration. Clin Experiment Ophthalmol 2009; 37:275-85; Da Cruz L, Chen F K, Ahmado A, Greenwood J, Coffey P. RPE transplantation and its role in retinal disease. Prog Retin Eye Res 2007; 26:598-635]. While graft survival is observed in some cases, improvements in visual acuity are disappointing to date [Berson E L, Jakobiec F A. Neural retinal cell transplantation: Ideal versus reality. Ophthalmology 1999; 106:445-46].
Experimental research suggests that stem cell transplantation shows promise for reconstituting the damaged cellular populations of the retina [Klassen H, Sakaguchi D S, Young M J. Stem cells and retinal repair. Prog Retin Eye Res 2004; 23:149-81; Enzmann V, Yolcu E, Kaplan H J, Ildstad S T. Stem cells as tools in regenerative therapy for retinal degeneration. Arch Ophthalmol 2009; 127:563-71]. One of the key advantages of using stem cells is their potential to differentiate into any type of cell, including retinal neurons and RPE [Das A M, Zhao X Ahmad I. Stem cell therapy for retinal degeneration: Retinal neurons from heterologous sources. Semin Ophthalmol 2005; 20:3-10]. For cell replacement therapy in the retina, the discovery of adult retinal stem cells (RSCs) [Tropepe V, Coles B L K, Chiasson B J, Horsford D J, Elia A J, McInnes R R et al. Retinal stem cells in the adult mammalian eye. Science 2000; 287:2032-36] and their isolation in humans [Coles B L K, Angenieux B, Inoue T, Rio-Tsonis K, Spence J R, McInnes R R et al. Facile isolation and the characterization of human retinal stem cells. Proc Natl Acad Sci USA 2004; 101:15772-7] was a major step forward, avoiding the ethical concerns regarding the use of embryonic/fetal tissue [Sugarman J. Human Stem Cell Ethics: Beyond the Embryo. Cell Stem Cell 2008; 2:529-33]. It has been shown that cells derived from the pigmented ciliary margin can give rise to all retinal cell types as well as can integrate into the retinae of early postnatal mice [Coles B L K, Angenieux B, Inoue T, Rio-Tsonis K, Spence J R, McInnes R R et al. Facile isolation and the characterization of human retinal stem cells. Proc Natl Acad Sci USA2004; 101:15772-7]. The developing mouse eye is a permissive environment for cellular integration due to the presence of differentiation and proliferation signals and the absence of a mature glial limitans membrane, which prevents transplanted cells from migrating into the neural retina in adult intravitreal cellular transplantation [Kinouchi R, Takeda M, Yang L, Wilhelmsson U, Lundkvist A, Pekny M et al. Robust neural integration from retinal transplants in mice deficient in GFAP and vimentin. Nat Neurosci 2003; 6:863-8]. To bypass this membrane in adults, the target for cellular replacement therapy is subretinal. Barriers to adult subretinal transplantation include cellular survival and host tissue integration. It has been well documented that cell death, leakage and migration from the injection site occurs when retinal progenitor cells are delivered as a single cell suspension in saline [Klassen H J, Ng T F, Kurimoto Y, Kirov I, Shatos M, Coffey P et al. Multipotent retinal progenitors express developmental markers, differentiate into retinal neurons, and preserve light-mediated behavior. Invest Ophthalmol V is Sci 2004; 45:4167-73].
To overcome the poor survival and tissue integration associated with subretinal delivery, retinal progenitor cells have been delivered to the retina on solid biomaterial scaffolds [Redenti S, Neeley W L, Rompani S, Saigal S, Yang J, Klassen H et al. Engineering retinal progenitor cell and scrollable poly(glycerol-sebacate) composites for expansion and subretinal transplantation. Biomaterials 2009; 30:3405-14; Redenti S, Tao S, Yang J, Gu P, Klassen H, Saigal S et al. Retinal tissue engineering using mouse retinal progenitor cells and a novel biodegradable, thin-film poly(ecaprolactone) nanowire scaffold. J Ocul Biol Dis Infor 2008; pp 1-11 In press; Neeley W L, Redenti S, Klassen H, Tao S, Desai T, Young M J et al. A microfabricated scaffold for retinal progenitor cell grafting. Biomaterials 2008; 29:418-26; Tad. S, Young C, Redenti S, Zhang Y, Klassen H, Desai T et al. Survival, Migration and differentiation of retinal progenitor cells transplanted on micro-machined poly(methyl methacrylate) scaffolds to the subretinal space. Lab Chip 2007; 7:695-701; Tomita M, Lavik E, Klassen H, Zahir T, Langer R, Young M J. Biodegradable polymer composite grafts promote the survival and differentiation of retinal progenitor cells. Stem Cells 2005; 23:1579-88]. These tissue engineered porous scaffolds are composed of common synthetic polymers including poly(L-lactic acid)/poly(lactic-co-glycolic acid) (PLLA/PLGA) [Lavik E B, Klassen H, Warfvinge K, Langer R, Young M J. Fabrication of degradable polymer scaffolds to direct the integration and differentiation of retinal progenitors. Biomaterials 2005; 26:3187-96], poly(methyl methacrylate) (PMMA) [Tao S, Young C, Redenti S, Zhang Y, Klassen H, Desai T et al. Survival, migration and differentiation of retinal progenitor cells transplanted on micro-machined poly(methyl methacrylate) scaffolds to the subretinal space. Lab Chip 2007; 7:695-701], poly(ε-caprolactone) (PCL) [Redenti S, Tao S, Yang J, Gu P, Klassen Saigal S et al. Retinal tissue engineering using mouse retinal progenitor cells and a novel biodegradable, thin-film poly(ecaprolactone) nanowire scaffold. J Ocul Biol Dis Infor 2008; 1:19-29], or poly(glycerol-sebacate) (PGS) [Redenti S, Neeley W L, Rompani S, Saigal S, Yang J, Klassen H et al. Engineering retinal progenitor cell and scrollable poly(glycerol-sebacate) composites for expansion and subretinal transplantation. Biomaterials 2009; 30:3405-14]. They are often coated with laminin to enhance cell adhesion and penetration into the porous polymer scaffold. While important advances have been made, these solid scaffolds do not match the modulus of the retina and lack the flexibility required for subretinal delivery across the damaged retina [Tomita M, Lavik E, Klassen H, Zahir T, Langer R, Young M J. Biodegradable polymer composite grafts promote the survival and differentiation of retinal progenitor cells. Stem Cells 2005; 23:1579-88].
Efficient cell delivery and survival are major barriers to successful cellular transplantation. Most transplanted cells die, and those that remain viable either migrate away from the transplant site and/or aggregate together and thus do not integrate with the host tissue [Klassen H J, Ng T F, Kurimoto Y, Kirov I, Shatos M, Coffey P et al. Multipotent retinal progenitors express developmental markers, differentiate into retinal neurons, and preserve light-mediated behavior. Invest Ophthalmol V is Sci 2004; 45:4167-73].